Part one, the MHC-I aspect of this project, is directed to understand the fine details of the CD8 T cell response, its time course and specificity. Using several vectors expressing an HIV-1 envelope we showed that CD8 cells were elicited with different fine specificities and kinetics of mobilization. Several variant peptides of the immunodominant peptide were shown to bind the MHC-I restricting element, H-2Dd better than the parent envelope-derived peptide, and T cell clones with distinct specificities were elicited by the different vectors. High resolution X-ray crystal structures revealed major differences in solvent exposure of epitopic residues of the peptide antigen, consistent with the different populations of T cells. These findings suggest that different gene-based vectors generate peptides with alternate conformations within MHC-I that elicit distinct T cell responses after vaccination. The second part of this project is focused on functional studies of T cell receptor recognition of autoantigens and how this leads to autoimmune disease. Our current approaches include: 1) the characterization of T cell receptors derived from autoreactive T cell clones, functionally and structurally;2) the development of transgenic mouse strains that constitutively express a limited TCR repertoire consisting of autoreactive T cell receptors;3) the functional characterization of these autoreactive TCR transgenic mouse strains;4) the characterization of the antigenic peptides recognized by the TCR transgenic mouse strains both functionally and biochemically;and 5) the in vivo characterization of processing of autoantigens as this process relates to T lymphocyte selection and development of autoimmune disease. We have cloned and expressed two different TCR from T cell clones that show specificity for two peptides from the gastric H/K ATPase. On transfer to immunodeficient animals, one of these clones causes a Th1 type disease, and the other a Th2-like disease. The Th2-like disease is characterized by T cells producing IL4, IL5, and IL13 and shows leukocyte infiltrates in the gastric mucosa. Transgenic animals expressing the TCR from each of these clones have been produced and have been analyzed. The transgenic derived from the Th1 clone, TXA23, develops a fulminant autoimmune gastritis within 10 days of birth. The transgenic derived from the Th2 clone, TXA51, has a less agressive disease. This second model offers to provide insight into how inflammatory (Th1) cytokines influence autoimmune disease in a manner distinct from Th2 cytokines. Cells taken from the Th2 diseased animals can be maintained in vitro as Th2 cells, or if stimulated with progressive doses of antigenic peptide presented by dendritic cells, can differentiate into Th1 cells. Several hypotheses concerning the differential cytokine and disease profiles of the two transgenic strains have been investigated: 1) is the intrinsic affinity of the TXA23 TCR greater for its MHC/peptide complexthan that of the TXA51 TCR;and 2) is the efficiency of the processing and presentation of the antigenic peptide seen by TXA23 better than that of the peptide seen by TXA51. Careful functional dose-response experiments and IAd/peptide tetramer staining experiments are consistent with the view that the TXA51 TCR affinity for peptide/MHC is greater than that of TXA23 TCR. Cell transfer experiments indicate that TXA23 CD4 T cells transferred to normal mice proliferate extensively in the gastric lymph node while TXA51 cells fail to proliferate. Thus, the simplest explanation is that the affinity differences do not explain the functional phenomena, and that antigen processing and presentation differences do. This view is confirmed by quantitative measures of the stability of the antigenic peptide/MHC complex and will be further examined by direct measurement of the binding of engineered TXA23 and TXA51 TCR for the MHC/peptide complexes. We have determined the high resolution X-ray structure of the complex of the MHC-II molecule, IAd, in complex with the antigenic peptide that is recognized by the TXA51 mouse strain. Details of the organization of the peptide as bound to the MHC molecule reflect biochemical parameters of the strength of the molecular interaction as well as the autoimmune phenotype of the TXA51 transgenic animals.